The invention relates to novel ninjurin proteins, nucleic acids and antibodies.
A distinguishing characteristic of the peripheral nervous system (PNS), as opposed to the central nervous system (CNS), is its capacity for axonal regeneration after injury. The environment in which PNS axons regenerate consists of Schwann cells and their basal laminae, fibroblasts, collagen, degenerating myelin, and phagocytic cells (Fawcett, J. W. and Keynes, R. J. (1990). Annu. Rev. Neurosci. 13, 43-60; Bunge, R. and Griffin, J. W. (1992). The cell of Schwann. In Diseases of the Nervous system:Clinical Neurology. A. K. Asbury, G. M. McKhann, and W. I. McDonald, eds. (Philadelphia: W B Saunders), pp. 87-100). Among these components, Schwann cells are indispensable for axonal regeneration, as evidenced by the reduction in axonal growth when live Schwann cells are removed from the area of injury (Hall, S. M. (1986). Neuropathol. Appl. Neurobiol. 12, 401-414). Conversely, when transplants consisting of cultured Schwann cells and their associated extracellular matrix are introduced into a lesion in the CNS, axonal regeneration and subsequent re-innervation is facilitated, clearly indicating the unique role of Schwann cells in promoting axonal regeneration (Aguayo, A. J. (1985). Axonal regeneration from injured neurons in the adult mammalian central nervous system. In Synaptic plasticity and Remodeling. C. W. Cotman, ed. (New York: Guilford Press), pp. 457-483; Benfey et al., Nature 296, 150-152; Richardson et al., (1980). Nature 284, 264-265).
The interruption of the axon following nerve injury initiates a complex series of changes in the injured nerve. Rapid changes in the synthesis of myelin components occur, such as marked decreases in myelin lipid synthesis (Whiten et al., (1989) J. Neurochem. 52, 1085-1092) and diminished expression of the major myelin proteins (Trapp et al., (1988) J. Neurosci. 8, 3515-3521). Within three days after axotomy, Schwann cells in the distal stump begin to proliferate in a longitudinal band along which axonal regeneration and re-growth are most frequently observed (Chaudhry et al, (1992) Neurologic Clinics 10, 613-627). There are also a number of Schwann cell proteins whose expression is increased in the distal stump after nerve injury, including cell surface molecules like the p75 NGF receptor (Taniuchi et al., (1988) J. Neurosci. 8, 664-681), and cell adhesion molecules L1, N-cadherin and N-CAM, that are important for neurite outgrowth on Schwann cells in vitro (Martini et al., (1988) J. Cell. Biol. 106, 1746; Bixby et al., (1988) J. Cell. Biol. 107, 353-361; Rieger et al., (1988) J. Cell; Biol. 107, 707-719). Schwann cells also increase the expression of a number of diffusible molecules, including the neurotrophic factors, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), as well as apolipoprotein D, which may be involved in the recycling of cholesterol released from degenerating myelin (Spreyer et al, (1990) EMBO J 9, 2479-2484).
Most of the changes in Schwann cell phenotype in response to nerve injury appear to be dependent on the axon itself, as many of them are reversed as the axon regenerates. For instance, p75 expression is down-regulated in Schwann cells adjacent to the regenerating axon (Taniuchi et al., 1988) and in Schwann cells co-cultured with neurons (Tomaselli et al., (1986) J. Cell Biol. 103, 2659-2672; Fallon, J. R. (1985) J. Neurosci. 5, 3169-3177). In contrast, the expression of myelin proteins such as P0 and PMP-22 increases as the axon regenerates and remyelination ensues (White et al., 1989; Snipes et al., (1992) J. Cell Biol. 117, 225-238). Most of the molecules that mediate alterations in Schwann cell gene expression remain obscure; one exception is the increased expression of NGF that is mediated by IL-1 elaborated by macrophages that invade the lesioned nerve (Lindholm et al., (1987) Nature 330, 658-659).
Cell surface adhesion proteins have also been shown to play a role in tissue regeneration after injury in a number of organisms. Cell surface adhesion proteins also play an important role in embryonic development and in the assembly of adult organs. In vertebrates, a number of cell surface glycoproteins have been identified as adhesion molecules, including integrins, cadherins, and those containing a immunoglobulin(Ig)-like motif.
The role of adhesion proteins in nerve regeneration has been documented (Martini, R. (1994) J. Neurocytology 23, 1-28; Brodkey et al., (1993) Exp. Neurol. 123, 251-270). The expression of a number of adhesion proteins is elevated after nerve injury, including N-CAM and L1, which are thought to be involved in forming a suitable substrate for the extension of the regenerating axons. The time course of this up-regulation has been reported (Daniloff et al., (1986) J. Cell Biol. 103, 929-945; Martini, 1994). Interestingly, the levels of N-CAM and L1 each return to normal more rapidly after a crush injury than after transection, in accord with the more rapid and complete recovery observed after nerve crush (Daniloff et al., 1986). Direct support for the role of adhesion proteins in promoting neurite outgrowth has come from the demonstration that neutralizing antibodies to L1 and N-cadherin inhibit Schwann cell stimulated neurite outgrowth from peripheral motor neurons (Seilheimer et al., J. Cell Biol. 107, 341-351). However, these studies also suggest that additional molecules are also important, as no single antibody or combination of antibodies was capable of totally eliminating process outgrowth. In the case of L1 and N-cadherin, homophilic interactions between molecules present on neuronal outgrowths and Schwann cells are likely to be responsible for their growth promoting effects (Lemmon et al., (1989) Neuron 2, 1597-1603; Takeichi, M. (1991) Science 251, 1451-1455).
The residues which mediate the adhesive interactions of these molecules have been identified for only a subset of these proteins. One of the most well-characterized sequence motifs of this type is the tripeptide Arg-Gly-Asp (RGD) which was identified as the sequence within fibronectin that mediates cell attachment. Many integrins recognize this RGD motif within their respective ligands, and these interactions then mediate either cell-substratum or cell-cell interactions.
Members of the cadherin family contain multiple copies of the sequences, Asp-Arg-Glu (DRE) and Asp-x-Asn-Asp-Asn (DXNDN (SEQ ID NO:1)) sequences. Structural analysis of cadherin indicates that these motifs may be situated such that they can form a zipper-like structure that may be critical for cell adhesion.
Shared sequence motifs for members of the Ig-superfamily of adhesion molecules have not been reported, although it has been proposed that a decapeptide sequence (KYSFNYDGSE (SEQ ID NO: 2)) in the third Ig-like domain of neural cell adhesion molecule (NCAM) is responsible for its homophilic binding interactions
It is an object of the present invention to provide novel proteins which play a role in axonal regeneration of PNS neuronal cells after injury. Accordingly, the invention provides recombinant ninjurin proteins and variants thereof, and to produce useful quantities of these ninjurin proteins using recombinant DNA techniques.
It is a further object of the invention to provide recombinant nucleic acids encoding ninjurin proteins, and expression vectors and host cells containing the nucleic acid encoding the ninjurin protein.
An additional object of the invention is to provide polyclonal and monoclonal antibodies directed against ninjurin proteins.
A further object of the invention is to provide methods for producing the ninjurin proteins.
In accordance with the objects outlined above, the present invention provides recombinant nucleic acids encoding ninjurin proteins, including fragments of the full-length proteins, and ninjurin peptides. The ninjurin proteins include ninjurin 1 and ninjurin 2. The ninjurin peptides include variant or derivative ninjurin peptides.
In a further aspect, the invention provides expression vectors comprising transcriptional and translational regulatory DNA operably linked to DNA encoding a ninjurin protein, and host cells containing the expression vectors.
In an additional aspect, the invention provides methods for producing ninjurin proteins comprising the steps of culturing a host cell transformed with an expressing vector comprising a nucleic acid encoding a ninjurin protein and expressing the nucleic acid to produce a ninjurin protein.
In a further aspect, the invention provides recombinant ninjurin proteins including fragments of the full-length proteins, and ninjurin peptides, including derivative ninjurin peptides.
In an additional aspect, the invention provides pharmaceutical compositions comprising ninjurin proteins, and polypeptides capable of specifically binding to a ninjurin protein, including antibodies.
In a further aspect, the invention provides polyclonal or monoclonal antibodies to ninjurin proteins.
In an additional aspect, the invention provides METHODS OF TREATMENT, ETC.?